Compositions and methods for treatment of staphylococcal infection while suppressing formation of antibiotic-resistant strains

ABSTRACT

Co-administration of a lysostaphin or other anti-staphylococcal agent which cleaves cross-links of peptidoglycans of staphylococci cell walls such as lysostaphin and an antibiotic effective against staphylococci due to antibiotic activity mediated by cell-wall activity is effective against staphylococcal infection, even staphylococci that may be resistant to one or other of lysostaphin or the cell-wall active antibiotic. Co-administration simultaneously suppresses the generation of antibiotic-resistant mutant strains. Effective cell-wall active antibiotics include β-lactams and glycopeptides.

This invention was made with the support of the United States Governmentunder STTR Grant Number R41-HL60334-01, which was awarded by theNational Institutes of Health. The United States Government may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to a method of treating staphylococcal infectionin mammals, including humans. The method involves the simultaneousadministration of a lysostaphin or other agent which attacks theglycine-containing peptide cross-links of the cell wall peptidoglycanfound in staphylococci and an antibiotic, the antibiotic properties ofwhich are mediated by its ability to affect the cell wall of the targetstaphylococci. This combined administration is effective in treating thestaphylococcal infection, and at the same time suppresses the formationof strains resistant to lysostaphin or other peptidoglycan active agent.

2. Background of the Prior Art

Lysostaphin is a bacteriocin secreted by a staphylococcal strainisolated and originally named Staphylococcus staphylolyticus (now S.simulans). The production oflysostaphin is described in U.S. Pat. No.3,278,378. Lysostaphin is an endopeptidase which cleaves the polyglycinecross-links of the peptidoglycan found in the cell walls ofstaphylococci. U.S. Pat. Nos. 3,398,056 and 3,594,284 describeimprovements to culture medium and inoculation techniques for theproduction of lysostaphin.

The gene for lysostaphin from S. simulans has been sequenced and cloned,U.S. Pat. No. 4,931,390. Lysostaphin for use as a laboratory reagent hasbeen produced by fermentation of a non-pathogenic recombinant strain ofB. sphaericus, from which it is readily purified. The cloning andsequencing of the lysostaphin gene permits the isolation of variantenzymes that have properties similar to or different from those of wildtype lysostaphin. One such altered enzyme, bearing a single amino acidchange, has been characterized and shown to have potentanti-staphylococcal activity both in vitro and in an animal infectionmodel. U.S. patent application Ser. No. 09/120,030, filed Jul. 21,1998and incorporated herein by reference. Other lysostaphin analogues,including naturally occurring enzymes of this type have been establishedas potent agents capable of addressing difficult to treat bacterialdiseases caused by staphylococcal infection. Other peptidases withrelated activity are known. Thus lasA protease and achromopeptidase,reported in Kessler. et al., J. Biol. Chem. 268:7503-08 (1993) and Li etal., J. Biochem. 122:772-778(1997), respectively, haveanti-staphylococcal activity based on their digestion ofglycine-containing cross-links in the peptidoglycan cell wall component.These agents may be used in this invention in place of lysostaphin.

The development of lysostaphin as an effective antibiotic to treatstaphylococcal infection has been plagued, however, by a problem that isuniversal for antibiotic administration—the increasing development ofantibiotic-resistant strains of mutant staphylococci. Already, a widevariety of staphylococcal infections resistant to various antibioticsthat were previously the treatment of choice, including methicillin(methicillin resistant S. aureus are referred to as MRSA) andvancomycin-resistant strains (referred to as VISA) have been identified.Resistance to a wide variety of other antibiotics, not exhibited bysensitive staphylococci, has been noted as well. MRSA, as well asstrains resistant to other antibiotics, are discussed at length inStranden, et al., J. Bacteriology 179(1):9-16 (1997). Furtherdifficulties are encountered in that MRSA tend to accumulate a varietyof other resistances as well. Multiresistant MRSA are typically treatedwith vancomycin, The Staphylococci In Human Diseases, 158-174 (Grossley,et al., editors 1997). Vancomycin itself may be toxic. Additionally,vancomycin resistance has recently been detected in staphylococciinfections.

The problem posed by the continuing development of antibiotic-resistantinfectious agents, such as staphylococci, is more than the difficultyinvolved in treating any individual patient. Popular press, as well asscientific journals, have noted the alarming increase in the generationof resistant strains, due in part to indiscriminate use or over-use ofantibiotics. Each time an individual is treated with an antibiotic,whether needlessly or reasonably, the chance that a strain resistant tothat particular treatment will arise is increased. Resistant strains ofstaphylococci have become endemic in many hospitals and pose alife-threatening danger to patients already debilitated by otherailments who become infected after admission to those hospitals.

Numerous articles have noted the development of resistance to eitherlysostaphin or β-lactams, such as methicillin, and the relationshipthere between. Thus, DeHart, et al., Applied Environmental Microbiology61:1475-1479 (1995) noted the development of mutant S. aureusrecombinant cells that were resistant to lysostaphin, but susceptible tomethicillin. Similar phenomenon are reported by Zygmunt, et al., Can. J.Microbio. 13:845-853 (1967), Polak, et al., Diagn. Microbiol. Infect.Dis. 17:265-270 (1993) and Dixon, et al., Yale J. Bio. Med. 41:62-67(1968). Each of these references, as well as later reports such asEhlert, J. Bacteriology 179:7573-7576 (1997), note that staphylococcithat develop resistance to lysostaphin, either spontaneously or throughinduced recombination, become susceptible to methicillin treatment, andvice-versa. In all of these references, the uniform suggestion is tofollow a course of administration of lysostaphin, even a short one, withadministration of methicillin.

U.S. Pat. No. 5,760,026, commonly assigned herewith, employs a specificmethod for treating mastitis, by intramammary infusion of lysostaphin.The patent reports, Table ID and elsewhere, that a synergistic result ispredicted when combining lysostaphin and a β-lactam to treat mastitis,based on an in vitro assay. The bovine mastitis model is not predictiveof in vivo administration of antibiotics, and the synergistic effectsreported in U.S. Pat. No. 5,760,026 have not been substantiated in anenvironment or model that would be reflective of in vivo administrationto a mammal such as a human.

Those of skill in the art will be aware that there are a wide variety ofstaphylococcal strains. Many are resistant to conventional antibiotics,unlike sensitive strains. S. aureus strains are recognized as highlyvirulent and the most common single cause of serious systemicinfections. Coagulase-negative staphylococcal species, althoughgenerally less invasive than S. aureus, are now responsible for asignificant incidence of infections; particulary among debilitated orimmunocompromised patients. As an example of such infection, one maypoint to endocarditis consequent to heart valve replacement. This is butone of a variety of intractable staphylococcal infections which areincreasing due to the widespread use of antibiotics.

Accordingly, it remains an object of those of ordinary skill in the artto develop a method whereby even resistant staphylococcal infections inmammals, including humans, may be effectively treated by theadministration of antibiotics. Desirably, this method is developed so assuppress the formation of strains resistant to the antibiotics used.

SUMMARY OF THE INVENTION

The above goals, and others made clear by the discussions set forthbelow, are achieved by the simultaneous administration of ananti-staphylococcal agent, such as lysostaphin or other agent whoseactivity is mediated by cleavage of glycine-containing cross-links inthe staphylococcal cell wall peptidoglycan and an antibiotic orantimicrobial agent whose activity is mediated by its ability to affectthe cell wall of staphylococci. These cell-wall active agents includeβ-lactams and glycopeptides. Preferably, the cell-wall active antibioticis a β-lactam.

There is no evidence of any synergistic effect achieved through thesimultaneous administration of an anti-staphyloccocal agent whoseactivity is mediated by cleavage of glycine-containing cross-links and acell-wall active antibiotic in a model, in vitro or in vivo, that ispredictive of benefit for in vivo administration of antibiotics in amammal. Indeed, those of ordinary skill in the art will recognize thatfor resistant staphylococci, such as MRSA, the administration ofmethicillin is not therapeutically effective in any amount.Surprisingly, Applicants have discovered that the combinedadministration of an anti-staphyloccocal agent whose activity ismediated by cleavage of glycine-containing cross-links such aslysostaphin and the cell-wall active antibiotic not only effectivelytreats the infection, but suppresses the formation of staphylococcihaving resistance to the anti-staphylococcal agent whose activity ismediated by cleavage of glycine-containing cross-links.

While Applicants do not wish to be bound by this explanation, it appearsthat the spontaneous mutation commonly effective in conferringlysostaphin resistance in staphylococci renders the same highlysusceptible to a cell-wall active antibiotic, such as methicillin. Thisis true even where the organism starts out as methicillin resistant.Simultaneous administration of both appears to be uniformly effective insimultaneously eradicating the infection and suppressing the generationof new resistant strains. Specifically, anti-staphylococcal agents likelysostaphin cleave glycine-containing cross-links. The mutationconferring resistance to this attack renders previously resistantstrains sensitive to cell wall active antibiotics.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the administration of a pharmaceuticalcomposition effective in the treatment of staphylococcal infection,which composition comprises at least two active agents, one an agentlike lysostaphin which cleaves the glycine-containing cross-links of thecell wall peptidoglycans of staphylococci, the other a cell-wall activeantibiotic. By lysostaphin it is intended to refer herein to any enzyme,including lysostaphin wild type, a mutant or variant, or any recombinantor related enzyme that retains proteolytic activity againstglycine-containing cross-links in the cell wall peptidoglycan ofstaphylococci. Variants may be generated by post-translationalprocessing of the protein (either by enzymes present in a producerstrain or by means of enzymes or reagents introduced at any stage of theprocess) or by mutation of the structural gene. Mutations may includesite-deletion, insertion, domain removal and replacement mutations. Theymay be recombinantly expressed, or otherwise. Other anti-staphylococcalactive agents acting by cleavage of the glycine-containing peptidoglycancross-links include lasA protease and achromopeptidase. Suchanti-staphylococcal agents which affect the peptidoglycan cross-linksare embraced by the invention, but exemplified herein by reference tolysostaphin.

Cell-wall active antibiotics include β-lactams and glycopeptides.β-lactams are preferred. Suitable β-lactams include, but are not limitedto, penicillins, such as penicillin, nafcillin, oxacillin, methicillin,amoxicillin and cloxacillin. Other β-lactams include cephalosporins andcarbapenems. Representative cephalosporins include cephalothin,cefazolin, cefamandole, ceftazidime and others. Suitable carbapenemsinclude imepenem and meropenem.

Suitable glycopeptides include vancomycin, teicoplanin and ramoplanin.

These two agents can be combined with further agents, adjuvants and thelike, but are effectively administered in a pharmaceutically acceptablecarrier. Administration is typically systemic, and may be intravenous(IV), intramuscular (IM), subcutaneous (SC), intraperitoneal (IP),intrathecal or topical. No synergistic effect of combining lysostaphinand a β-lactam or glycopeptide or cell-wall active antibiotic has beennoted in a model predictive of in vivo mammalian administration.Accordingly, each agent of the effective combination must beadministered in a therapeutically effective amount. It is to be noted,in this regard, that the amount to be administered is that which istherapeutically effective when the lysostaphin and cell-wall activeagent are administered together. Those of skill in the art will ofcourse recognize that there is no therapeutically effective amount for,e.g.,methicillin if the staphylococcal infection is an MRSA infection.Nonetheless, administration of therapeutic amounts of methicillin asdetermined against non-MRSA, combined with an amount of lysostaphineffective against staphylococci that are not lysostaphin-resistant willeffectively treat staphylococcal infections even where the infection isresistant to one or other antibiotic. Accordingly, applicants havereferred herein to “therapeutically effective amounts” to mean amountseffective to therapeutically treat sensitive S. aureus infection. Thissimultaneous administration, as opposed to sequential administrationtypified by the prior art, also surprisingly results in the suppressionof strains resistant against either antibiotic, or their combination.

Any of a wide variety of pharmaceutically acceptable carriers anddiluents, typically buffered, may be used. Appropriate pharmaceuticalcarriers are known to those of skill in the art. The formulations ofthis invention comprise a therapeutic amount of lysostaphin and atherapeutic amount of a cell-wall active antibiotic, such that whenco-administered, the staphylococcal infection, either S. aureus orcoagulase negative staphylococci, is treated, while the generation ofresistant strains is suppressed. Other active agents that do notinterfere with the activity of the two antibiotics may beco-administered.

Therapeutic values will range substantially given the nature of thestaphylococcal infection, the individual, and the antibiotic being usedin conjunction with lysostaphin. Representative values foranti-staphyloccocal active agents such as lysostaphin, range fromapproximately 15-150 mg/kg body weight/day for human administration,with a preferred range of 25-100 mg/kg/day. Values for β-lactams basedon nafcillin range from 50-250 mg/kg/day, with a preferred range of100-220 mg/kg/day and glycopeptides like vancomycin are administeredover a range of 10-75 mg/kg/day, with a preferred range of 15-50mg/kg/day.

The administration course is not substantially different from thatcurrently administered in single antibiotic treatments, and can rangefrom 7-28 days, although typically, courses of 7-21 days are employed,and effective in treating a wide variety of staphylococcal infections.

EXAMPLES

To compare the development of resistant strains, growth curves for threemethicillin resistant staphylococcal strains were obtained for in vitrogrowth in Mueller Hinton Broth.

Growth curves were completed in Mueller Hinton Broth (50 ml) in glasserlenmeyer flasks. Flasks were inoculated with 100 μl of an overnightgrowth adjusted to 0.5 Macfarland to yield a starting concentration ofapproximately 10⁵-10⁶ CFU/ml. Growth curves were done in the presence oflysostaphin, lysostaphin and oxacillin (1 μg/ml) or no antibiotics(controls). Absorbance at OD 600 was recorded at 0, 2, 4, 6 and 24hours. At 24 hours flasks were plated on MHA, MHA with lysostaphin (6μg/ml) and MHA with oxacillin (6 μg/ml) in order to screen for resistantmutants. Three methicillin resistant Staphylococcus aureus strains weretested: 272855, 450M and Mu3.

Growth following 24 hour incubation with lysostaphin (0.0625 μg/ml),lysostaphin (0.0625 μg/ml) and oxacillin (1 μg/ml), and no antibiotics,was recorded.

The data generated led to the following conclusions:

1. The addition of oxacillin to lysostaphin led to significantsuppression of growth for all three strains.

2. The presence of oxacillin suppressed the expression of lysostaphinresistance among all three strains.

In order to demonstrate the effectiveness of the claimed invention,certain experiments were conducted. Checkerboard susceptibility testingwas conducted to determine whether simultaneous administration oflysostaphin and oxacillin (a β-lactam) would be effective in suppressingthe development of resistance. Oxacillin concentrations varied between0.0156 μg/ml and 1 μg/ml. Lysostaphin concentrations varied between0.00048 and 0.9 μg/ml. Four strains were tested for evidence of synergybetween lysostaphin and oxacillin; 27619, Col, 27227 and VA348. Therewas no evidence of synergy or antagonism over the concentration rangetested. The MIC of lysostaphin was unchanged in the presence ofoxacillin in concentrations up to 1 μg/ml for all strains tested. Theovernight growth of strains in the presence of lysostaphin and oxacillinwas examined. Four strains were grown overnight in drug free media(MHB), MHB with 0.1 μg/ml of lysostaphin, MHB with lysostaphin 0.1 μg/mland oxacillin 1 μg/ml, and MHB with oxacillin 1 μg/ml. The four strainstested included 450M, Col, and their lysostaphin resistant mutants 450 Mlyso and Col lyso. The results are reflected in Table 1.

TABLE 1 Growth in the presence of lysostaphin 0.1 μg/ml oxacillin Lyso +oxacillin Isolates MHB alone μg/ml combn 450M + + + − 450M lyso + + − −Col + + + − Col lyso + + − −

The same unpredicted result has been demonstrated through in vivoexperiments based on the widely accepted rabbit model of aortic valveendocarditis, predictive of in vivo administration to humans. Whenadministered to staphylococcal infected rabbits at low doses (1 mg/kgbid, as compared with a minimum value of 5 mg/kg tid for sterilization),lysostaphin, as representative of anti-staphylococcal agents acting bycleavage of the glycine-containing cross-links, resulted in recovery ofa number of resistant colonies, with high counts in vegetations andkidneys, while the same dosage together with nafcillin (a β-lactam) gavesterile kidneys, some sterile vegetations, and no resistant strainsrecovered. The simultaneous treatment of staphylococcal infection withsuppression of resistant strain formation is an exciting and widelyuseful invention nowhere predicted in the art. This invention offers thepossibility of treating staphylococcal infections while suppressing thegeneration of strains resistant to any or all active agentsadministered.

The inventive compositions and methods of this application have beendisclosed generically, and by reference to specific example, examplesare not intended to be limiting unless so indicated, and variations willoccur to those of ordinary skill in the art without the exercise ofinventive faculty. In particular, variations in the identity of thecell-wall active antibiotic to be co-administered with ananti-staphylococcal agent acting by cleavage of the glycine-containingcross-links, as well as various recombinant and mutant variants thereof,carriers and concentrations will occur to those of skill in the artwithout the exercise of inventive faculty, and remain within the scopeof the invention, unless specifically excluded by the claims set forthbelow.

What is claimed is:
 1. A pharmaceutical composition in dosage form fortreating a staphylococcal infection in a human subject, said compositioncomprising: lysostaphin in an amount of from 15 to 150 mg/kg body weightof the human subject; and a β-lactam antibiotic in an amount of from 50to 250 mg/kg body weight of the human subject, wherein said composition,when administered to the human subject for a period of time sufficientto eradicate said infection, suppresses formation of staphylococcalstrains resistant to said lysostaphin, said cell-wall active antibioticand said composition, and wherein said amount of lysostaphin is anamount effective in treating, in a human, a staphylococcal infectionthat is not lysostaphin-resistant and wherein said amount of thecell-wall active antibiotic is an amount effective in treating, in ahuman, a staphylococcal infection that is not resistant to the cell-wallactive antibiotic.
 2. The composition of claim 1, wherein the β-lactamis selected from the group consisting of a penicillin, a cepalosporinand a carbapenem.
 3. The composition of claim 2, wherein the β-lactam ispenicillin.
 4. The composition of claim 1, wherein the β-lactam isnafcillin.
 5. The composition of claim 4, wherein the compositioncomprises nafcillin in an amount of from 100 to 200 mg/kg body weight ofthe human subject.
 6. The composition of claim 1, wherein the β-lactamis a cephalosporin selected from the group consisting of cephalothin,cefazolin, cefamandole, and ceftazidime.
 7. The composition of claim 1,wherein the β-lactam is a carbapenem selected from the group consistingof imepenem and meropenem.
 8. The composition of claim 1, wherein theβ-lactam is oxacillin.
 9. The composition of claim 1, further comprisinga pharmaceutically acceptable carrier.
 10. The composition of claim 1,wherein the composition is in a form suitable for intravenous,intramuscular, subcutaneous, intraperitoneal, intrathecal or topicaladministration.